Mechanismof Diisopropylamine Dichloroacetate Enhancing The Radiosensitizationof Esophageal Squamous Cell Carcinoma

Section I Bioinformatics analysis of esophageal squamous cell carcinoma genomeBackground:At present,esophageal carcinoma is the sixth leading cause of cancer death in the world and is one of the most common gastrointestinal tumors merely after gastric cancer,rectum cancer and liver cancer in China.With approximately two hundred thousand patients dying of esophageal carcinoma every year,China accounts for nearly half of the incidences.As life science steps into the post-genome era,interaction of life science,information science,microelectronics and computer science enables the continuous breakthrough in high-throughput chips and next-generation sequencing,which leads to the lower costs and the new concept of transcriptomics in developments of cancer.Methods:Data collection GSE92396 were downloaded from the Gene Expression Omnibus(GEO)and processed with the online analyze tool GE02R to analyze the discriminating expression levelsbetween tumor and adjacent normal tissues.CCLE(Cancer Cell Line Encyclopedia)was applied to screen the genes which expressed differently in tumor and normal tissues and then validated by our own tissues.Results:1789differentially expressed genes were screened out through the data collection GSE92396.Among them,907 genes were high-expressed and 882 were low-expressed in tumor.Those genes were also validated in squamous cell carcinoma cells TE9?KYSE510?TE6?KYSE520 by CCLE and our own squamous cancer tissues.Conclusion:Development and progpression of esophageal carcinoma may be associated with PI3K-Akt pathways,glycolysis,constitution of extracellular matrix,and cell adhesion.Section ? Effect of Diisopropylamine dichloroacetate on radiosensitizationin esophageal carcinomaBackground:Radiotherapy(RT)is recommended as the primarytreatment modality for esophageal carcinoma.However,the radio-resistance of esophageal carcinoma remains an obstacle for the effective treatment of esophageal carcinoma.Enhanced glycolysis is a common trait of manytypes of human cancers.Consistent with many types of solid tumors,esophageal carcinoma is highly glycolytic,producing largeamounts of lactic acid as a metabolic by-product.Accumulated evidence supports the notion that glycolysisis associated with radio-resistance of esophageal carcinoma.By consulting literature we found that diisopropylamine dichloroacetate could operate the cell metabolism and thus reverse the glycolysis.Methods:We applied CCK8assay to comparetheantiproliferative effectof DCA and DADA in esophageal carcinoma.Matrigel?Transwell assays were used to evaluate the effect of DADA on invasion and metastasisof esophageal carcinoma cells.PT staining flow cytometry and western blot were used to assess the activated cleaved caspase-3 in order to evaluate the impact of DADA on esophageal carcinomaapoptosis.Results:CCK8 assay revealed that DADA exhibited stronger antiproliferative effect on esophageal carcinoma cell line Eca-109and TE-13 than DCA.Matrigeland Transwell assay indicated that DADA could inhibitinvasion and metastasis of esophageal carcinoma.Results of flow cytometry and western blot indicated that apoptosis of esophageal carcinoma cells elevated after treatment of DADA.Conclusion:These results showed that the DADA itself could exert anti-tumor fuctionSection ?Mechanism of Diisopropylamine dichloroacetate enhancing radiosensitizationin esophageal carcinomaBackground:The radiation energy andthe ROS produced from intercellular water caused abreakdown of double-stranded DNA and directly damagedcellular proteins.The indirect effect is the secondaryresponse involved in gene expression and cell signalingby the second messenger ROS.It is proven that glycolysis was associated with radiosensitization and the messenger ROS.DADA embodies the antitumor activity and plays a role in regulating the cell metabolism and glycolysis.Methods:By using clone formation assay we evaluated the impact of DADA on radiotherapy sensitization.We also took advantage of western blotting to measure the protein levels of y-H2AX after radiotherapy,DADA treatment and the combination treatment.The XF Extracellular Flux Analyzer(Seahorse Bioscience)was used to measure the oxygen consumption rate(OCR).Animal experiments were approved by the EthicsCommittee of Nanjing Cancer Hospital.A Reactive Oxygen Species Assay kit(KeyGEN)was used to detect the intracellular levels of ROSResults:The combination of DADA and radiotherapy could significantly reduce the formation of esophageal cancer cells cloned group.To matching the survival curve,we found that DADA can enhance the radiation sensitivity of Eca-109 and TE-13,increased by 1.37 folds and 1.37 folds respectively.Testing the cells after exposure to radiotherapy and DADA with immunofluorescence and western blot,we found the expression of y-H2AX significantly higher.Flow cytometry is used to test cancer cells treated with DADA along with radiotherapy,the result showed that cells blocked in G2/M phase and]apoptosis rate increased.In vivo experiments showed that,compared with single radiotherapy group or DADA group,the Joint of DADA and radiotherapy could inhibit the growth of nude mice subcutaneous tumor.Mechanism research has shown that,the Joint of DADA and radiotherapy could Significantly increased the ROS level in the esophageal cancer cell lines.To test oxygen consumption rate in the cell with XF Extracellular Flux Analyzer,we could found that the oxygen consumption rate of TE-13 and Eca-109 obviously increased and the level of cell oxidative phosphorylation elevated after treated with DADA.Conclusion:In our present study,we found thatDADA could modulate oxidative phosphorylation as well as increase the intracellularlevels of reactive oxygen species(ROS).